The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sela, U.
Right arrow Articles by Lider, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sela, U.
Right arrow Articles by Lider, O.
The Journal of Immunology, 2005, 175: 7255-7263.
Copyright © 2005 by The American Association of Immunologists

The Inhibition of Autoreactive T Cell Functions by a Peptide Based on the CDR1 of an Anti-DNA Autoantibody Is via TGF-{beta}-Mediated Suppression of LFA-1 and CD44 Expression and Function1

Uri Sela*,{dagger}, Nora Mauermann*, Rami Hershkoviz{dagger}, Heidy Zinger*, Molly Dayan*, Liora Cahalon*, Jian Ping Liu*, Edna Mozes2,* and Ofer Lider3,*

* Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and {dagger} Assaf-Harofeh Medical Center and Sakler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE), which is characterized by the increased production of autoantibodies and defective T cell responses, can be induced in mice by immunization with a human anti-DNA mAb that expresses a major Id, designated 16/6Id. A peptide based on the sequence of the CDR1 of the 16/6Id (human CDR1 (hCDR1)) ameliorated the clinical manifestations of SLE and down-regulated, ex vivo, the 16/6Id-induced T cell proliferation. In this study, we examined the mechanism responsible for the hCDR1-induced modulation of T cell functions related to the pathogenesis of SLE. We found that injection of hCDR1 into BALB/c mice concomitant with their immunization with 16/6Id resulted in a marked elevation of TGF-{beta} secretion 10 days later. Addition of TGF-{beta} suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGF{beta}-induced inhibition of T cell proliferation is by down-regulating the expression, and therefore the functions, of a pair of key cell adhesion receptors, LFA-1 ({alpha}L{beta}2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-{beta}-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1{alpha} and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE-associated responses by hCDR1 treatment may be due to the effect of the up-regulated TGF-{beta} on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)4 is an autoimmune disease characterized by increased production of autoantibodies and defective T cell-mediated responses. The latter are associated with diverse clinical manifestations that involve multiple organ systems (1). Experimental SLE can be induced in naive, non-SLE-prone mice by their immunization with anti-DNA mAbs that express the major Id, designated 16/6Id, of either human or mouse origin (2, 3). The 16/6Id-induced disease in mice resembles SLE in humans, because it is manifested by high levels of autoantibodies (anti-dsDNA and anti-nuclear protein Ab) and by SLE-associated clinical symptoms. Peptides based on the sequences of the CDR1 and CDR3 of the murine 16/6Id mAb and on the CDR1 of the human 16/6Id were synthesized and characterized. The latter were shown to down-regulate in vitro and in vivo autoreactive T cell responses and to ameliorate the clinical manifestations of spontaneous and 16/6Id-induced SLE in mice (4, 5, 6, 7, 8). The beneficial effects of the treatment with the CDR-based peptides were associated with a down-regulation of IFN-{gamma}, IL-10, and TNF-{alpha} secretion and an up-regulation of the immunosuppressive cytokine TGF-{beta} (5, 6, 8).

T cells play an important role in the pathogenesis of lupus (9, 10, 11, 12), and their proliferation and expansion were reported to correlate with the severity of lupus (13). Therefore, down-regulation of pathogenic T cell functions, such as adhesion, migration, and proliferation, and, as a result, the burden of autoreactive T cells is of major importance in ameliorating the disease activity. Indeed, we have shown recently that treatment with human CDR1 (hCDR1) concomitant with 16/6Id immunization down-regulates stromal cell-derived factor-1{alpha} (SDF-1{alpha}; CXCL12)-induced adhesion and migration both early after immunization (10 days) and when mice with full-blown lupus are treated with hCDR1 (14).

Classically, two signals are needed for Ag-specific, APC-associated T cell activation and proliferation: 1) interaction of the TCR with MHC class II molecules loaded with a peptide, and 2) interaction of the costimulatory molecule CD28 with B7 (15). However, it is also well established that other costimulatory molecules participate in this process (16, 17, 18), of which, LFA-1 (CD11a/CD18) and CD44 have been found to be up-regulated and activated in lupus (19, 20, 21, 22). The latter receptors also participate in trafficking and recruitment of activated T cells into the site of inflammation through cell-cell and/or cell-matrix interaction (22). The blockade or down-regulation of these adhesion and costimulatory molecules has been shown to be beneficial in various autoimmune diseases, including lupus (23, 24, 25, 26).

In the present study, we assessed the putative importance of CD44 and LFA-1 in the early stages after immunization of mice with the 16/6Id and concomitant treatment with hCDR1. We report in this study that suppression of the 16/6Id-specific T cell proliferation by hCDR1 involves the inhibition of LFA1 and CD44 expression and their adhesive function in vitro. These effects are mediated by hCDR1-induced up-regulation of TGF-{beta} secretion, because the latter cytokine by itself down-regulates T cell proliferation and LFA-1 and CD44 expression and function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mice of the BALB/c inbred strain were obtained from Harlan. Female mice were used at 8–10 wk of age.

Reagents

The following reagents and chemicals were purchased from the sources indicated: RPMI 1640, Invitrogen Life Technologies; FCS, HEPES buffer, antibiotics, and sodium pyruvate, Kibbutz Beit-Haemek; recombinant human SDF-1{alpha}, R&D Systems; PMA and hyaluronic acid (HA), Sigma-Aldrich; and recombinant mouse ICAM-1/Fc chimera, R&D Systems. Recombinant hTGF-{beta}1 was obtained from PeproTech. Na251[Cr]O4 was purchased from Amersham Biosciences. PD98059 (an ERK inhibitor) was obtained from Calbiochem. IFN-{gamma} was purchased from BD Pharmingen.

Synthetic peptide

A peptide based on the sequence of the CDR1 of the human mAb anti-DNA that bears the major 16/6Id Id (GYYWSWIRQPPGKGEEWIG) was synthesized by Polypeptide Laboratories using solid phase synthesis by F-moc chemistry. The hCDR1 (Edratide) is under clinical development for the treatment of SLE by TEVA Pharmaceutical Industries. As a control, we used a peptide containing the amino acids of hCDR1 (SKGIPQYGGWPWEGWRYEI) in a scrambled order (designated scrambled peptide). This peptide binds MHC class II with an avidity similar to that of hCDR1 (M. Dayan and E. Mozes, unpublished observations).

Abs and mAb

The human anti-DNA mAb that bears the 16/6Id (IgG1/{kappa}) was previously described (27). The Ab is secreted by hybridoma cells that are grown in culture and purified by using a protein G-Sepharose column (Pharmacia). Rat anti-mouse CD11a (integrin {alpha} L chain, LFA-1 {alpha}-chain) mAb and rat anti-mouse CD44 mAb (clone IM7) were purchased from BD Pharmingen. Their isotype controls were purchased from the sources indicated: rat IgG2a (Serotec) and rat IgG2b (Southern Biotechnology Associates), respectively.

Immunization with 16/6Id, hIgG, or keyhole limpet hemocyanin (KLH) and treatment with hCDR1

Mice were immunized with 1 µg of the human mAb 16/6Id in CFA intradermally into the hind foot pads. An additional group of mice was injected s.c. with the hCDR1 (50 µg/mouse) or the control scrambled peptide concomitant with 16/6Id immunization. Additional control groups of mice were similarly immunized with hIgG or KLH (Calbiochem) in CFA and either treated or not with the hCDR1. Mice of all groups were killed on day 10, and their lymph node (LN)-derived T cells were studied.

T cells

Preparation of an enriched population of LN-derived T cells was performed as follows: petri dishes were precoated (overnight, 4°C) with goat anti-mouse Ig (15 µg/ml in 5 ml of PBS) and then washed three times. Inguinal murine LN cells were incubated (70 min, 4°C, in RPMI 1640 containing 10% FCS and antibiotics) on the coated plates. The nonadherent cells, which were mainly T cells (>92%, as assessed by FACS analysis), were then collected and washed in RPMI 1640.

T cell adhesion assays

Analysis of T cell adhesion to immobilized protein substrates was determined as previously described (28, 29). Briefly, flat-bottom microtiter plates were precoated with either ICAM-1 or HA in PBS (2.4 and 50 µg/ml, respectively), and the remaining binding sites were blocked with PBS containing 1% BSA. Next, the purified murine LN-derived T cells were labeled with 51Cr and resuspended in RPMI 1640 medium supplemented with 1% HEPES buffer and 0.1% BSA (adhesion medium) in the presence of SDF-1{alpha} (100 ng/ml) or PMA (50 ng/ml). The plates were incubated (60 min, 37°C, in a 7.5% CO2 humidified atmosphere) and then gently washed. The adherent cells were lysed (H2O containing 1 M NaOH and 0.1% Triton X-100), collected, and counted by a gamma counter (Packard Instruments). The results (±SD) are expressed as the mean percentage of bound T cells of quadruplicate wells.

Stimulation and detection of TGF-{beta}1

LN-derived cells were taken from mice 10 days after immunization with the 16/6Id mAb or hIgG and concomitant treatment with hCDR1 or the control peptide. Cells (5 x 106) were incubated in the presence of 16/6Id (25 µg/ml) or hIgG, and their supernatants were collected after 72 h. The levels of TGF-{beta}1 were measured by ELISA according to the manufacturer’s instructions, using recombinant hTGF-{beta}1 (sRII/Fc chimera; R&D Systems). Supernatants were added after activation of latent TGF-{beta}1 to immunoreactive TGF-{beta}1 by adding HCl and then neutralizing the acidified sample with NaOH (according to the manufacturer’s instructions). A standard recombinant TGF-{beta}1 was used as well. A biotinylated anti-hTGF-{beta}1 (R&D Systems) was used as the secondary Ab. The assay was developed using tetramethylbenzidine color reagent (Helix Diagnostix), and enzyme activity was evaluated using 570- and 630-nm filters.

FACS assay

T cells (106 cells/sample) were incubated (30 min, 4°C) with FITC-conjugated mAb rat anti-mouse CD11a (Serotec) and rat anti-mouse CD44 (Southern Biotechnology Associates). The cells were then washed, resuspended in FACS buffer (PBS containing 0.05% sodium azide; final concentration, 5 x 105 cells in 0·5 ml), and analyzed by FACSort (BD Biosciences). The reduction in CD11a or CD44 expression was calculated as follows: % reduction = [(% of stained cells of the immunized mice) – (% of stained cells of the immunized and hCDR1 treated mice)] x 100/% of stained cells of the immunized mice.

Proliferation assays

Ten days after immunization with 16/6Id, hIgG, or KLH, mice were killed and inguinal LNs were used for proliferation assays. Cells (1 x 106) were cultured in the presence of the immunizing agent (0.1–10 µg/well) or medium only. Cultures were set up (in triplicate) in 200 µl of RPMI 1640-enriched medium containing 1% syngeneic normal mouse serum in 96-well microtiter plates (Nunc). After 96 h, [3H]thymidine was added to the wells. Eighteen hours later, the plates were harvested onto a filter plate. Incorporation of [3H]thymidine was measured using a beta counter.

Statistical analysis

The nonparametric Mann-Whitney U test was used for statistical analyses of the data. A value of p ≤ 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
hCDR1 down-regulates 16/6Id-specific proliferation by inhibition of LFA-1 and CD44

The inhibitory effect of hCDR1 is first presented by its ability to inhibit a 16/6Id-specific proliferation response. Fig. 1A shows that immunization of BALB/c mice with 16/6Id and concomitant treatment with hCDR1 significantly down-regulated the proliferation response stimulated by 16/6Id. Table I demonstrates that the inhibitory effect of hCDR1 is specific, because a control peptide (scrambled) did not inhibit proliferation of T cells from 16/6Id-immunized mice. To further assess the specific effect of hCDR1, mice were immunized with Ag other than 16/6Id, namely, general hIgG (because 16/6Id is a specific hIgG Ab) and the nonrelated Ag KLH. As shown in Table I, the hCDR1 did not down-regulate hIgG- or KLH-stimulated proliferation in vitro.



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 1. In vivo treatment with hCDR1 or in vitro addition of anti-LFA-1 or anti-CD44 mAbs down-regulated 16/6Id-specific proliferation. T cells were purified from LN of 16/6Id-immunized BALB/c mice that were either not treated or were concomitantly treated with hCDR1 (A). LN cells were obtained from BALB/c mice immunized with 16/6Id, and the cells were incubated in vitro with mAb anti-LFA-1 (B) or anti-CD44 (C) or with their isotype controls. 16/6Id (at various concentrations (A) or 5 µg/well (B and C)) was added to the cultures. After 96 h, [3H]thymidine was added to the wells. Eighteen hours later, plates were harvested onto a filter plate, and incorporation of [3H]thymidine was measured using a beta counter. Depicted are the results of one experiment representative of three, which yielded similar results. *, p < 0.05.

 

View this table:
[in this window]
[in a new window]
  		Table I. hCDR1 inhibits specifically the proliferation to 16/6Ida

 
LFA-1 and CD44 are adhesion and costimulatory molecules that are up-regulated in lupus. To determine whether these molecules may affect the 16/6Id-triggered proliferation similarly as treatment with hCDR1, we studied the effect of blocking LFA-1 or CD44 on the proliferation of LN cells derived from mice immunized 10 days previously with 16/6Id. The cells were incubated with mAb anti-LFA-1 (50 µg/ml) or -CD44 (10 µg/ml) and stimulated with 16/6Id. LFA-1 (Fig. 1B) or CD44 (Fig. 1C)-specific mAb, but not their isotype controls, inhibited 16/6Id-induced proliferation to levels comparable to those found after treatment with hCDR1 (Fig. 1A).

To find out whether treatment with hCDR1 affects the expression of LFA-1 and CD44, we stained purified T cells with Abs to these receptors and analyzed the results by FACS. As shown in Fig. 2A1, the majority of the T cells derived from 16/6Id-immunized mice that were either untreated or concomitantly treated with hCDR1 expressed levels of LFA-1 with a log fluorescence intensity <100. A small subset of cells expressed significantly higher levels (log fluorescence intensity, 100-1000) of this receptor. The inhibitory effect (32% reduction) of treatment with hCDR1 was mainly on this relatively small population of cells with the higher expression levels of LFA-1. An enlargement of the area of log fluorescence intensity 100-1000 (the area under the M1 in Fig. 2A1) is shown in Fig. 2A2. The cells in this high log fluorescence intensity region might represent the 16/6Id-activated T cells that proliferate in vitro in response to 16/6Id, because a 30% reduction in LFA-1 expression was observed in CD45RBhigh-stained T cells derived from hCDR1-treated mice (Fig. 2C). Similarly, as shown in Fig. 2B1, treatment with hCDR1 lowered the expression levels of CD44 mainly (35%) on T cells expressing high levels of CD44 (log fluorescence intensity, 100-1000; see enlargement in Fig. 2B2). When gated to CD45RBhigh-stained T cells, a 28% reduction in CD44 expression was observed (Fig. 2D). Thus, treatment with hCDR1 down-regulates the expression of LFA-1 and CD44, affecting more prominently the cells with high expression levels of these two adhesion and costimulatory molecules. The effect of hCDR1 on the expression of LFA-1 and CD44 was determined to be specific, because the control peptide did not down-regulate the latter (LFA-1 expression was 33, 34, and 22.5%; CD44 expression was 16.4, 16.5, and 11.5% for 16/6Id-immunized, 16/6Id and control peptide treatment, and 16/6Id and hCDR1 treatment, respectively). In addition, hCDR1 did not decrease the expression of these receptors when given as treatment to mice immunized with hIgG (LFA-1 expression was 27 vs 26.8%; CD44 expression was 15.3 vs 15.7% for hIgG-immunized and hIgG plus hCDR1 treatment, respectively).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2. Treatment with hCDR1 down-regulates LFA-1 and CD44 expression mainly on activated T cells derived from 16/6Id-immunized mice. T cells were purified from LN of 16/6Id-immunized mice that either were not treated or were treated with hCDR1. The cells were incubated with anti-LFA-1 (A1) or anti-CD44 (B1) Abs and analyzed using FACSort. A2 and B2 are enlargements of the area of log fluorescence intensity 100-1000, M1 in A1 and B1, respectively (1 denotes T cells derived from 16/6Id-immunized mice, 2 denotes T cells derived from 16/6Id-immunized and hCDR1-treated mice). The expression of these two receptors was also measured on the subpopulation of CD45RBhigh-stained T cells (C and D, respectively). Depicted are the results of one experiment, which is representative of five that yielded similar results. The percent reduction in receptor expression (calculated as described in Materials and Methods) was statistically significant (p < 0.05).

 
Next, we examined whether the apparent reduced expression of LFA-1 and CD44 by hCDR1 affected their binding to their respective ligands, ICAM-1 and HA, respectively, and therefore may account for the inhibition of proliferation. To this end, we studied the adhesion of T cells isolated from mice immunized with 16/6Id and treated with hCDR1 to these ligands. Fig. 3, which depicts the results of one representative experiment of three performed, shows that treatment of mice with hCDR1 resulted in a significant (p < 0.001) down-regulation of SDF-1{alpha}- and PMA-induced T cell adhesion to ICAM-1 and HA, respectively. As demonstrated for proliferation, a control peptide did not down-regulate SDF-1{alpha}- and PMA-induced T cell adhesion to ICAM-1 and HA, and hCDR1 did not affect adhesion of T cells derived from hIgG-immunized mice to these ligands. Thus, the hCDR1-induced decrease in LFA-1 and CD44 expression on T cells derived from 16/6Id-immunized mice is associated with a pronounced down-regulation in the ability of these cells to bind their ligands.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. Treatment of BALB/c mice with hCDR1 concomitant with 16/6Id immunization down-regulated the ex vivo SDF-1{alpha}- and PMA-induced T cell adhesion to ICAM-1 and HA. Labeled T cells from 16/6Id- or hIgG-immunized and hCDR1- or control peptide-treated mice were added to ICAM-1 (A) or HA (B)-coated microtiter well plates and were incubated with or without 100 ng/ml SDF-1{alpha} or 50 ng/ml PMA. The wells were washed 60 (A) or 150 (B) min later, and the adhesion of the remaining ICAM-1 or HA-bound T cells, respectively, was then measured. Shown are the results ± SD of one experiment that is representative of three performed. *, p < 0.05.

 
hCDR1 up-regulates TGF-{beta}, which inhibits 16/6Id-stimulated proliferation by down-regulating LFA-1 and CD44

It was of interest to determine whether the ability of hCDR1 to down-regulate SLE-associated responses, including proliferative responses to 16/6Id is via the up-regulation of TGF-{beta} secretion. Fig. 4A shows that treatment with hCDR1 up-regulated TGF-{beta} secretion by LN cells derived from 16/6Id-immunized mice (considered 100% = 280 pg/ml). It should be noted that the in vivo treatment with hCDR1 up-regulated TGF-{beta} secretion also in the absence of 16/6Id stimulation in vitro, but to a lesser extent (160% = 448 pg/ml vs 210% = 587 pg/ml in the absence or the presence of 16/6Id stimulation in vitro, respectively). The specificity of the treatment with hCDR1 was determined by the inability of the hCDR1 to up-regulate TGF-{beta} secretion by LN cells derived from hIgG-immunized mice. Furthermore, levels of TGF-{beta} secretion did not increased after treatment of 16/6Id-immunized mice with the control peptide.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4. Treatment with hCDR1 concomitant with 16/6Id immunization up-regulates specifically TGF-{beta} secretion that down-regulates 16/6Id-stimulated LN cell proliferation. A, LN were removed from BALB/c mice that were immunized 10 days previously with 16/6Id or hIgG and either not treated or concomitantly treated with hCDR1 or control peptide. Cells (5 x 106/ml) were incubated with 16/6Id (25 µg/ml) for 72 h. TGF-{beta} was determined in the cell supernatants by ELISA. Results are expressed as the percentage of TGF-{beta} levels in supernatants of cells derived from 16/6Id-immunized mice and in vitro stimulated with 16/6Id (100% = 280 pg/ml). B, LN cells of mice immunized with 16/6Id were incubated with the indicated concentrations of TGF-{beta} and stimulated with 16/6Id. C, LN cells obtained from mice treated with hCDR1 concomitant with their immunization with 16/6Id were incubated in vitro with anti-TGF-{beta} mAb or with an isotype control and were stimulated with 16/6Id. [3H]Thymidine was added after 96 h of incubation (B and C). Depicted are the results of one experiment that is representative of three yielding similar results. *, p < 0.05.

 
The role of TGF-{beta} in inhibiting the proliferative response is demonstrated in Fig. 4B; exogenous addition of TGF-{beta} in vitro (250 and 1000 pg/ml) to LN cells derived from mice that were immunized with 16/6Id significantly down-regulated the 16/6Id-specific proliferation (Fig. 4B). This figure also shows that the extent of inhibition by TGF-{beta} was comparable to that by hCDR1.

To confirm the role of TGF-{beta} in the inhibitory effect of hCDR1 on in vitro LN cell proliferation, we incubated LN cells derived from hCDR1-treated mice with an anti-TGF-{beta} Ab and stimulated them with 16/6Id. As shown in Fig. 4C, the TGF-{beta}-specific mAb, but not its isotype control, markedly abrogated the suppression of 16/6Id-triggered LN cell proliferation after in vivo treatment with hCDR1. In fact, LN cell proliferation after treatment with anti-TGF-{beta} Ab was similar to that of 16/6Id-immunized murine LN cells. Thus, inhibition of the proliferation of LN cells derived from hCDR1-treated mice is due, at least partially, to the elevated levels of TGF-{beta} after this treatment.

In contrast to the up-regulation of TGF-{beta} by the hCDR1, the latter down-regulates the secretion of the pathogenic cytokine IFN-{gamma} (8). To determine the contribution of the decrease in IFN-{gamma} secretion to the inhibition of proliferation, IFN-{gamma} (500 pg/ml) was added to LN cells derived from 16/6Id-immunized and hCDR1-treated mice. The results indicated that IFN-{gamma} abrogated the inhibitory effect of hCDR1 on 16/6Id-stimulated proliferation (3,682 and 10,016 cpm for LN cells from 16/6Id-immunized and hCDR1-treated mice, in the absence or the presence of IFN-{gamma}, respectively, compared with 7,951 cpm for LN cells from 16/6Id-immunized mice).

To determine whether secreted TGF-{beta} that is up-regulated by hCDR1 may affect the expression of the two adhesion and costimulatory molecules, namely, LFA-1 and CD44, we incubated T cells derived from 16/6Id-immunized mice with TGF-{beta} for 24 h. Cells were stained and analyzed by FACS to evaluate changes in receptor expression. In vitro incubation with TGF-{beta} (250 pg/ml) reduced the expression of these receptors by 33 and 28%, respectively, mainly on the small subpopulation of cells with high log fluorescence intensity (data not shown). This subpopulation of cells might represent the 16/6Id-activated T cells that proliferate in vitro in response to 16/6Id, because, as shown in Fig. 5, 38 and 26% reductions in LFA-1 and CD44 expression, respectively, were observed when gated to CD45RBhigh-stained T cells. Thus, TGF-{beta} down-regulates the expression of LFA-1 and CD44, affecting more prominently the cells with high expression levels of these two adhesion and costimulatory molecules.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 5. TGF-{beta} down-regulates LFA-1 and CD44 expression mainly on activated T cells derived from 16/6Id-immunized mice. T cells, purified from LN of 16/6Id-immunized mice, were incubated with TGF-{beta} (250 pg/ml) in vitro for 24 h. The cells were then incubated with anti-LFA-1 (A) or anti-CD44 (B) Abs to determine the expression of the latter molecules on a subpopulation of CD45RBhigh-stained T cells. Depicted are the results of one experiment, which is representative of three that yielded similar results. The percent reduction in receptor expression was statistically significant (p < 0.05).

 
To determine the effect of TGF-{beta} on the binding of the costimulatory molecules, LFA-1 and CD44, to their ligands, ICAM-1 and HA, respectively, we incubated T cells, isolated from mice 10 days after 16/6Id-immunization, with TGF-{beta} (250 pg/ml; 24 h) and analyzed their adhesion to these ligands. Fig. 6, A and B, depicting the results of one representative experiment of three performed, shows that in vitro incubation with TGF-{beta} resulted in a significant (p < 0.001) down-regulation of SDF-1{alpha}- and PMA-induced T cell adhesion to ICAM-1 and HA, as observed after treatment with hCDR1 (Fig. 3). Thus, TGF-{beta}-induced inhibition of LFA-1 and CD44 expression on T cells derived from 16/6Id-immunized mice is accompanied by a pronounced decrease in the ability of these cells to bind their respective ligands.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 6. TGF-{beta} down-regulates the SDF-1{alpha}- and PMA-induced T cell adhesion to ICAM-1 and HA. T cells were obtained from BALB/c mice, immunized 10 days previously with 16/6Id, and incubated with TGF-{beta} (250 pg/ml) in vitro for 24 h (A and B). In the same manner, T cells obtained from mice that were treated with hCDR1 concomitant with their immunization with 16/6Id were incubated (24 h) in vitro with anti-TGF-{beta} mAb or the isotype control (C and D). T cell adhesion to ICAM-1 (A and C) and HA (B and D)-coated wells was then measured as described above. Data are from one experiment that is representative of three. *, p < 0.05.

 
To confirm the central role of TGF-{beta} in regulating the inhibitory effect of hCDR1 on SDF-1{alpha}- and PMA-induced adhesion to ICAM-1 and HA, we incubated T cells derived from hCDR1-treated mice with anti-TGF-{beta} mAb (24 h) and then studied their adhesion to these ligands. Fig. 6, C and D, demonstrates that the TGF-{beta}-specific mAb, but not its isotype control, completely abrogated the inhibitory effect of hCDR1. The observed levels of T cell adhesion to ICAM-1 or HA after incubation of cells with anti-TGF-{beta} Ab were similar to those of 16/6Id-immunized murine T cells. Thus, the inhibition of adhesion of T cells derived from hCDR1-treated mice is apparently due to the elevated levels of TGF-{beta} after this treatment.

Inhibition of ERK down-regulates the expression and function of LFA-1 and CD44

We have previously shown (14) that both hCDR1 and TGF-{beta} down-regulate ERK phosphorylation. Therefore, it was of interest to find out whether this kinase affects the expression and function of LFA-1 and CD44. To this end, T cells obtained from 16/6Id-immunized mice were incubated with the ERK inhibitor, PD98059, for 24 h and double-stained for CD45RB and LFA-1 or CD44. As shown in Fig. 7, A and B, incubation with PD98059 down-regulated the expression of both receptors (32 and 24%, respectively). It is noteworthy that the decrease in expression was similar to that shown for hCDR1 and TGF-{beta} (Fig. 2, C and D, and Fig. 5, respectively).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 7. Inhibition of ERK phosphorylation down-regulates the expression and function of LFA-1 and CD44. T cells were obtained from BALB/c mice, immunized 10 days previously with 16/6Id, and incubated with the ERK inhibitor PD98059 in vitro for 24 h. The expression of LFA-1 (A) and CD44 (B) was then measured on the subpopulation of CD45RBhigh-stained T cells using FACS. In addition, T cell adhesion to ICAM-1 (C) and HA (D)-coated wells was measured as described (in Materials and Methods). *, p < 0.05.

 
To determine whether inhibition of ERK phosphorylation may affect the ability of the cells to adhere to the ligands of these two receptors, HA and ICAM-1, respectively, an adhesion assay was performed on T cells obtained from 16/6Id-immunized mice that were incubated with PD98059. As shown in Fig. 7, C and D, inhibition of ERK phosphorylation significantly down-regulated SDF-1{alpha}- and PMA-induced T cell adhesion to ICAM-1 and HA. Thus, inhibition of ERK down-regulates both the expression and function of LFA-1 and CD44.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The main findings of this study are that hCDR1 down-regulated specifically the expression and function of the two tested adhesion and costimulatory molecules, LFA-1 ({alpha}L{beta}2) and CD44, which are important for T cell-APC interaction during proliferation. These effects are mediated by hCDR1-induced up-regulation of TGF-{beta} secretion and inhibition of ERK phosphorylation. To the best of our knowledge, this is the first demonstration of a direct linkage between TGF-{beta} and the suppression of LFA-1- and CD44-related functions in T cells.

The central role played by T cells in lupus has been previously demonstrated (9, 10, 11, 12). Human CDR1 was shown to be beneficial in the prevention and treatment of lupus (8). This was associated with down-regulation of the pathogenic cytokines (IFN-{gamma} and IL-10), including the proinflammatory cytokines (TNF-{alpha} and IL-1{beta}), and with up-regulation of the immunosuppressive cytokine TGF-{beta} (8). We have recently shown (14) that by 10 days after immunization with 16/6Id, well before the appearance of serological and clinical manifestations of experimental lupus, the responses to the disease-associated chemokine, SDF-1{alpha}, by T cells derived from hCDR1-treated mice were down-regulated. Based on this observation, we conducted the present study 10 days after immunization with16/6Id and concomitant treatment with hCDR1 in an attempt to elucidate the mode of action of hCDR1.

Our results show that hCDR1 down-regulated 16/6Id-stimulated cell proliferation. Proliferation and expansion of autoreactive T cells were reported to be correlated with the severity of lupus (13). Therefore, it is of major importance to down-regulate pathogenic T cell functions, such as adhesion and proliferation, to ameliorate the disease activity. In addition to the classical two signals needed for Ag-induced T cell proliferation during its interaction with APC, other costimulatory molecules participate in this interaction. The expression of two of these, LFA-1 and CD44, was shown to be up-regulated in lupus and was correlated with disease activity (19, 20). LFA-1 on T cells interacts with ICAM-1 on APC, and a blockade of this interaction induced the remission of diabetes in NOD mice (23). CD44 was shown to support proliferation by apposition of protein kinases for the initiation of signaling via the TCR/CD3 complex (30). It interacts with HA in the extracellular matrix, but, in addition, HA expressed on dendritic cells plays an important role during Ag presentation (31). The absence of CD44 has been recently shown to ameliorate disease in Faslpr/lpr disease (32). In agreement, our findings demonstrate that these two adhesion and costimulatory molecules play important roles in our model, because blockade of these two costimulatory molecules, LFA-1 and CD44, down-regulated 16/6Id-stimulated cell proliferation (Fig. 1, B and C). The inhibitory effect was similar to that achieved by treatment with hCDR1 (Fig. 1A).

The inhibition of T cell proliferation obtained from 16/6Id-immunized mice treated with hCDR1 was accompanied by a minor down-regulation in the expression of LFA-1 and CD44 on the whole T cell population (Fig. 2, A1 and B1). Nevertheless, a more pronounced down-regulatory effect was observed on a small subpopulation of T cells expressing high levels of these receptors (Fig. 2, A2 and B2). This subpopulation probably represents activated cells that participate in the process of proliferation, because when gated to CD45RBhigh-stained T cells that represent activated cells (33), a similar decrease of ~30% in the expression of these receptors was determined (Fig. 2, C and D). It is noteworthy that although the effect of treatment with hCDR1 on LFA-1 and CD44 expression was mild, it was reproducible, statistically significant (p < 0.05), and specific to 16/6Id-primed T cells. In addition to the minor reduction in their expression, a significant down-regulation was observed in the function of LFA-1 and CD44, namely, inhibition of the SDF-1{alpha}- and PMA-induced adhesion of T cells to their ligands on APC, ICAM-1 and HA, respectively (Fig. 3). The inhibition of adhesion to ICAM-1 and HA is likely to be due to down-regulation of LFA-1 and CD44, because other ligands that may adhere to HA or ICAM-1 are present mainly on cells other than T cells (31, 34).

TGF-{beta} is an immunosuppressive cytokine (reviewed in Refs.35, 36, 37), and both constitutive and stimulated levels of TGF-{beta} were shown to be low in SLE patients, especially when disease was active (38). Treatment of SLE-prone mice, which led to up-regulation of TGF-{beta}, ameliorated the serological and clinical manifestations (5, 8, 39, 40). Furthermore, the inhibition of 16/6Id-stimulated proliferation by hCDR1 demonstrated in the present study was associated with up-regulation of TGF-{beta} secretion. Indeed, in vitro addition of TGF-{beta} inhibited the 16/6Id-stimulated cell proliferation to a similar extent as in vivo treatment with hCDR1 (Fig. 4B). The role of TGF-{beta} in the inhibitory activity of hCDR1 was confirmed by demonstrating that anti-TGF-{beta} abrogated the effect of hCDR1 treatment (Fig. 4C).

Similar to in vivo treatment with hCDR1, incubation of 16/6Id-immunized murine T cells with TGF-{beta} mildly down-regulated the expression of the two adhesion and costimulatory molecules, LFA-1 and CD44, mainly on a subpopulation gated to CD45RBhigh-stained T cells (Fig. 5). This small population of T cells represents Ag (16/6Id)-induced activated T cells, which are probably the cells participating in 16/6Id-stimulated T cell proliferation. An inverse correlation between TGF-{beta} and LFA-1 has been shown by Xiao et al. (41), who found that decreased LFA-1 was associated with up-regulation of TGF-{beta}. In addition, T cells from TGF-{beta}–/– mice, which were shown to develop lupus-like disease (42), expressed high levels of the activation marker LFA-1 (43). Indeed, we demonstrate in this study that the down-regulation in the expression of these receptors by TGF-{beta} was accompanied by inhibition of their function, because in vitro addition of TGF-{beta} suppressed the ability of the cells to adhere to their respective ligands, namely, ICAM-1 and HA (Fig. 6, A and B). In addition to other mechanisms attributed to down-regulation of proliferation by TGF-{beta} (e.g., modulation of IL-2-induced activation of the Jak-Stat pathway in T cells (44) and alterations in the expression of cyclin-dependent kinase inhibitors that modify the sensitivity to TGF-{beta} by lowering thresholds for a maximal mitogenic response (45)), we suggest a mechanism by which up-regulation of this cytokine induces down-regulation of the expression and function of a pair of adhesion and costimulatory molecules that participate in the T cell-APC interaction during proliferation.

We have recently shown (14) that both in vivo treatment with hCDR1 as well as an in vitro addition of TGF-{beta} to T cells derived from 16/6Id-immunized mice down-regulated the phosphorylation of ERK, a kinase that plays a key role in signaling pathways associated with cell activation and their subsequent adhesion and migration (46, 47, 48). We show in this study that a specific blockade of the ERK pathway with PD98059 mildly down-regulated the expression of both LFA-1 and CD44 on T cells obtained from 16/6Id-immunized mice, similarly to hCDR1 and TGF-{beta}. Furthermore, this inhibition of ERK phosphorylation also significantly down-regulated the function of these receptors, because it inhibited their ability to adhere to their ligands (Fig. 7, C and D). Indeed, Tanimura et al. (49) demonstrated that inhibition of the ERK pathway decreased the expression of CD44 in tumor cells and thereby inhibited their invasiveness. Weber et al. (47) reported the involvement of the ERK pathway in down-regulation of LFA-1 avidity and transendothelial migration induced by SDF-1{alpha} in leukocytes.

We have shown in the present study that the effect of hCDR1 is specific to cells derived from 16/6Id-immunized mice and is not a global immunosuppressive one. Thus, it down-regulated proliferation, LFA-1 and CD44 expression, and adhesion of T cells derived from mice immunized with the human 16/6Id mAb. In contrast, hCDR1 did not affect cells derived from mice immunized with hIgG or with the nonrelevant Ag, KLH. The up-regulation of TGF-{beta} secretion by hCDR1 was shown to be specific to cells derived from 16/6Id-immunized mice; therefore, although the immunosuppressive effect of TGF-{beta} is not specific, it is likely to be restricted to the microenvironment of the 16/6Id-specific T cells.

It appears that hCDR1 exerts its beneficial effects via various pathways. In addition to the up-regulation of TGF-{beta} secretion and its subsequent inhibitory effects, hCDR1 affects other cytokines (8). It down-regulated IFN-{gamma} secretion that contributes to the inhibition of T cell proliferation. Thus, when this cytokine was added to T cells derived from 16/6Id-immunized and hCDR1-treated mice, it up-regulated their 16/6Id-stimulated proliferation significantly. In addition to the effects of hCDR1 on the above-mentioned cytokines, it is noteworthy that in vivo treatment with hCDR1 induced up-regulation of CD4+CD25+ regulatory cells (6–6.9% without treatment to 10.2–12.8% after treatment with hCDR1, in different experiments). These regulatory cells were shown to play a key role in the mechanism of action of the hCDR1 (N. Mauermann and E. Mozes, unpublished observations).

Taken together, our findings suggest the existence of a mechanism by which hCDR1 exerts its specific inhibitory capacities in vivo. The apparent down-regulation of murine T cell adhesion and proliferation is most likely a consequence of the peptide-induced up-regulation of TGF-{beta} secretion. We postulate that when present in relatively high concentrations, this cytokine, through down-regulation of ERK phosphorylation, can decrease T cell expression of two key adhesion and costimulatory molecules, LFA-1 and CD44. Consequently, there are down-regulations of T cell stimulation, adhesion, and proliferation, which are correlated with the disease activity and severity and are vital for the maintenance of chronic inflammatory reactions. Our findings in this study are based on experiments performed 10 days after immunization with 16/6Id and treatment with hCDR1. Nevertheless, hCDR1 was shown to be beneficial in the treatment of mice with established SLE (8), and this was accompanied by up-regulation of TGF-{beta} secretion and down-regulation of T cell migration (14). Furthermore, our preliminary results showed that LFA-1 expression is down-regulated in mice with established SLE after treatment with hCDR1 (U. Sela and E. Mozes, unpublished observations). Therefore, the mechanism described in this study for down-regulation of autoreactive T cell proliferation is likely to play a role in the amelioration of SLE manifestations after treatment with hCDR1.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Minerva Foundation, funded by the Committee for Scientific Cooperation between Germany and Israel (to O.L.), and by TEVA Pharmaceutical Industries, Israel (to E.M.). Back

2 Address correspondence and reprint requests to Dr. Edna Mozes, Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: edna.mozes{at}weizmann.ac.il Back

3 Died July 12, 2004. Back

4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; FN, fibronectin; hCDR, human CDR; HA, hyaluronic acid; KLH, keyhole limpet hemocyanin; LN, lymph node; SDF-1{alpha}: stromal cell-derived factor-1{alpha}. Back

Received for publication February 28, 2005. Accepted for publication September 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hahn, B. H.. 1993. An overview of the pathogenesis of systemic lupus erythematosus. B. H. Hahn, and D. J. Wallace, eds. Dubois’ Lupus Erythematosus 69 Williams & Wilkins, Philadelphia.
  2. Mendlovic, S., S. Brocke, Y. Shoenfeld, M. Ben-Bassat, A. Meshorer, R. Bakimer, E. Mozes. 1988. Induction of a systemic lupus erythematosus-like disease in mice by a common human anti-DNA idiotype. Proc. Natl. Acad. Sci. USA 85: 2260-2264. [Abstract/Free Full Text]
  3. Waisman, A., S. Mendlovic, P. J. Ruiz, H. Zinger, A. Meshorer, E. Mozes. 1993. The role of the 16/6 idiotype network in the induction and manifestations of systemic lupus erythematosus. Int. Immunol. 5: 1293-1300. [Abstract/Free Full Text]
  4. Waisman, A., P. J. Ruiz, E. Israeli, E. Eilat, S. Konen-Waisman, H. Zinger, M. Dayan, E. Mozes. 1997. Modulation of murine systemic lupus erythematosus with peptides based on complementarity determining regions of a pathogenic anti-DNA monoclonal antibody. Proc. Natl. Acad. Sci. USA 94: 4620-4625. [Abstract/Free Full Text]
  5. Zinger, H., E. Eilat, A. Meshorer, E. Mozes. 2003. Peptides based on the complementarity-determining regions of a pathogenic autoantibody mitigate lupus manifestations of (NZB x NZW)F1 mice via active suppression. Int. Immunol. 15: 205-214. [Abstract/Free Full Text]
  6. Eilat, E., M. Dayan, H. Zinger, E. Mozes. 2001. The mechanism by which a peptide based on complementarity-determining region-1 of a pathogenic anti-DNA auto-Ab ameliorates experimental systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA 98: 1148-1153. [Abstract/Free Full Text]
  7. Eilat, E., H. Zinger, A. Nyska, E. Mozes. 2000. Prevention of systemic lupus erythematosus-like disease in (NZBxNZW)F1 mice by treating with CDR1- and CDR3-based peptides of a pathogenic autoantibody. J. Clin. Immunol. 20: 268-278. [Medline]
  8. Luger, D., M. Dayan, H. Zinger, J. P. Liu, E. Mozes. 2004. A Peptide based on the complementarity determining region 1 of a human monoclonal autoantibody ameliorates spontaneous and induced lupus manifestations in correlation with cytokine immunomodulation. J. Clin. Immunol. 24: 579-590. [Medline]
  9. Fricke, H., S. Mendlovic, M. Blank, Y. Shoenfeld, M. Ben-Bassat, E. Mozes. 1991. Idiotype specific T-cell lines inducing experimental systemic lupus erythematosus in mice. Immunology 73: 421-427. [Medline]
  10. Ruiz, P. J., H. Zinger, E. Mozes. 1996. Effect of injection of anti-CD4 and anti-CD8 monoclonal antibodies on the development of experimental systemic lupus erythematosus in mice. Cell. Immunol. 167: 30-37. [Medline]
  11. Connolly, K., J. R. Roubinian, D. Wofsy. 1992. Development of murine lupus in CD4-depleted NZB/NZW mice: sustained inhibition of residual CD4+ T cells is required to suppress autoimmunity. J. Immunol. 149: 3083-3088. [Abstract]
  12. Chesnutt, M. S., B. K. Finck, N. Killeen, M. K. Connolly, H. Goodman, D. Wofsy. 1998. Enhanced lymphoproliferation and diminished autoimmunity in CD4-deficient MRL/lpr mice. Clin. Immunol. Immunopathol. 87: 23-32. [Medline]
  13. Lang, T. J., P. Nguyen, J. C. Papadimitriou, C. S. Via. 2003. Increased severity of murine lupus in female mice is due to enhanced expansion of pathogenic T cells. J. Immunol. 171: 5795-5801. [Abstract/Free Full Text]
  14. Sela, U., R. Hershkoviz, L. Cahalon, O. Lider, E. Mozes. 2005. Down-regulation of stromal cell-derived factor-1{alpha}-induced T cell chemotaxis by a peptide based on the complementarity-determining region 1 of an anti-DNA autoantibody via up-regulation of TGF-{beta} secretion. J. Immunol. 174: 302-309. [Abstract/Free Full Text]
  15. Mueller, D. L.. 2000. T cells: a proliferation of costimulatory molecules. Curr. Biol. 10: R227-R230. [Medline]
  16. Ni, H. T., M. J. Deeths, W. Li, D. L. Mueller, M. F. Mescher. 1999. Signaling pathway activated by leukocytes function-associated Ag-1-dependent costimulation. J. Immunol. 162: 5183-5189. [Abstract/Free Full Text]
  17. Pollard, K. M., M. Arnush, P. Hultman, D. H. Kono. 2004. Costimulation requirements of induced murine systemic autoimmune disease. J. Immunol. 173: 5880-5887. [Abstract/Free Full Text]
  18. Bertram, E. M., W. Dawicki, T. H. Watts. 2004. Role of T cell costimulation in anti-viral immunity. Semin. Immunol. 16: 185-196. [Medline]
  19. Takeuchi, T., K. Amano, H. Sekine, J. Koide, T. Abe. 1993. Upregulated expression and function of integrin adhesive receptors in systemic lupus erythematosus patients with vasculitis. J. Clin. Invest. 92: 3008-3016.
  20. Estess, P., H. C. DeGrendele, V. Pascual, M. H. Siegelman. 1998. Functional activation of lymphocyte CD44 in peripheral blood is a marker of autoimmune disease activity. J. Clin. Invest. 102: 1173-1182. [Medline]
  21. Ishikawa, S., S. Akakura, M. Abe, K. Terashima, K. Chijiiwa, H. Nishimura, S. Hirose, T. Shirai. 1998. A subset of CD4+ T cells expressing early activation antigen CD69 in murine lupus: possible abnormal regulatory role for cytokine imbalance. J. Immunol. 161: 1267-1273. [Abstract/Free Full Text]
  22. Nandi, A., P. Estess, M. Siegelman. 2004. Bimolecular complex between rolling and firm adhesion receptors required for cell arrest: CD44 association with VLA-4 in T cell extravasation. Immunity 20: 455-465. [Medline]
  23. Bertry-Coussot, L., B. Lucas, C. Danel, L. Halbwachs-Mecarelli, J. F. Bach, L. Chatenoud, P. Lemarchand. 2002. Long term reversal of established autoimmunity upon transient blockade of LFA-1/intracellular adhesion molecule-1 pathway. J. Immunol. 168: 3641-3648. [Abstract/Free Full Text]
  24. Mikecz, K., K. Dennis, M. Shi, J. H. Kim. 1999. Modulation of hyaluronan receptor (CD44) function in vivo in a murine model of rheumatoid arthritis. Arthritis Rheum. 42: 659-668. [Medline]
  25. Mikecz, K., F. R. Brennan, J. H. Kim, T. T. Glant. 1995. Anti-CD44 treatment abrogates tissue oedema and leukocyte infiltration in murine arthritis. Nat. Med. 1: 558-563. [Medline]
  26. Kevil, C. G., M. J. Hicks, X. He, J. Zhang, C. M. Ballantyne, C. Raman, T. R. Schoeb, D. C. Bullard. 2004. Loss of LFA-1, but not Mac-1, protects MRL/MpJ-Faslpr mice from autoimmune disease. Am. J. Pathol. 165: 609-616. [Abstract/Free Full Text]
  27. Waisman, A., Y. Shoenfeld, M. Blank, P. J. Ruiz, E. Mozes. 1995. The pathogenic human monoclonal anti-DNA that induces experimental systemic lupus erythematosus in mice is encoded by a VH4 gene segment. Int. Immunol. 7: 689-696. [Abstract/Free Full Text]
  28. Ariel, A., E. J. Yavin, R. Hershkoviz, A. Avron, S. Franitza, I. Hardan, L. Cahalon, M. Fridkin, O. Lider. 1998. IL-2 induces T cell adherence to extracellular matrix: inhibition of adherence and migration by IL-2 peptides generated by leukocyte elastase. J. Immunol. 161: 2465-2472. [Abstract/Free Full Text]
  29. Cherry, L. K., K. S. Weber, L. B. Klickstein. 2001. A dominant Jurkat T cell mutation that inhibits LFA-1-mediated cell adhesion is associated with increased cell growth. J. Immunol. 167: 6171-6179. [Abstract/Free Full Text]
  30. Foger, N., R. Marhaba, M. Zoller. 2000. CD44 supports T cell proliferation and apoptosis by apposition of protein kinases. Eur. J. Immunol. 30: 2888-2899. [Medline]
  31. Mummert, M. E., D. Mumert, D. Edelbaum, F. Hui, H. Matsue, A. Takashima. 2002. Synthesis and surface expression of hyaluronan by dendritic cells and its potential role in antigen presentation. J. Immunol. 169: 4322-4331. [Abstract/Free Full Text]
  32. Weber, G. F.. 2004. The absence of CD44 ameliorates Faslpr/lpr disease. Autoimmunity 37: 1-8. [Medline]
  33. Morrissey, P. J., K. Charrier, S. Brrady. 1993. CD4+ cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combines immunodeficient mice: disease development is prevented by cotransfer of purified CD4+ T cells. J. Exp. Med. 178: 237-244. [Abstract/Free Full Text]
  34. Matthys, P., K. Vermeire, A. Billiau. 2001. Mac-1 myelopoiesis induced by CFA: a clue to the paradoxical effects of IFN-{gamma} in autoimmune disease models. Trends Immunol. 22: 367-371. [Medline]
  35. Marek, A., J. Brodzicki, A. Liberek, M. Korzon. 2002. TGF-{beta} (transforming growth factor-{beta}) in chronic inflammatory conditions: a new diagnostic and prognostic marker?. Med. Sci. Monit. 8: RA145-RA151. [Medline]
  36. Letterio, J. J., A. B. Roberts. 1998. Regulation of immune responses by TGF-{beta}. Annu. Rev. Immunol. 16: 137-161. [Medline]
  37. Dean, G. S., J. Tyrrell-Price, E. Crawley, D. A. Isenberg. 2000. Cytokines and systemic lupus erythematosus. Ann. Rheum. Dis. 59: 243-251. [Free Full Text]
  38. Ohtsuka, K., J. D. Gray, M. M. Stimmler, B. Toro, D. A. Horwitz. 1998. Decreased production of TGF-{beta} by lymphocytes from patients with systemic lupus erythematosus. J. Immunol. 160: 2539-2545. [Abstract/Free Full Text]
  39. Raz, E., J. Dudler, M. Lotz, S. M. Baird, C. C. Berry, R. A. Eisenberg, D. A. Carson. 1995. Modulation of disease activity in murine systemic lupus erythematosus by cytokine gene delivery. Lupus 4: 286-292. [Abstract/Free Full Text]
  40. Sato, M. N., P. Minoprio, S. Avrameas, T. Ternynck. 2000. Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms. Clin. Exp. Immunol. 119: 333-339. [Medline]
  41. Xiao, B. G., G. X. Zhang, F. D. Shi, C. G. Ma, H. Link. 1998. Decrease of LFA-1 is associated with up-regulation of TGF{beta} in CD4+ T cell clones derived from rats nasally tolerized against experimental autoimmune myasthenia gravis. Clin. Immunol. Immunopathol. 89: 196-204. [Medline]
  42. Yaswen, L., A. B. Kulkarni, T. Fredrickson, B. Mittleman, R. Schiffman, S. Payne, G. Longenecker, E. Mozes, S. Karlsson. 1996. Autoimmune manifestations in the transforming growth factor-{beta}1 knockout mouse. Blood 87: 1439-1445. [Abstract/Free Full Text]
  43. Bommireddy, R., V. Saxena, I. Ormsby, M. Yin, G. P. Boivin, G. F. Babcock, R. R. Singh, T. Doetschman. 2003. TGF-{beta}1 regulates lymphocyte homeostasis by preventing activation and subsequent apoptosis of peripheral lymphocytes. J. Immunol. 170: 4612-4622. [Abstract/Free Full Text]
  44. Bright, J. J., L. D. Kerr, S. Sriram. 1997. TGF-{beta} inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes. J. Immunol. 159: 175-183. [Abstract]
  45. Wolfraim, L. M., T. M. Walz, Z. James, T. Fernandez, J. J. Letterio. 2004. p21Cip1 and p27Kip1 act in synergy to alter the sensitivity of naive T cells to TGF-{beta}-mediated G1 arrest through modulation of IL-2 responsiveness. J. Immunol. 173: 3093-3102. [Abstract/Free Full Text]
  46. Boehme, S. A., S. K. Sullivan, P. D. Crowe, M. Santos, P. J. Conlon, P. Sriramarao, K. B. Bacon. 1999. Activation of mitogen-activated protein kinase regulates eotaxin-induced eosinophil migration. J. Immunol. 163: 1611-1618. [Abstract/Free Full Text]
  47. Weber, K. S., G. Ostermann, A. Zernecke, A. Schroder, L. B. Klickstein, C. Weber. 2001. Dual role of H-Ras in regulation of lymphocyte function antigen-1 activity by stromal cell-derived factor-1{alpha}: implications for leukocyte transmigration. Mol. Biol. Cell 12: 3074-3086. [Abstract/Free Full Text]
  48. Woo, C. H., M. H. Yoo., H. J. You, S. H. Cho, Y. C. Mun, C. M. Seong, J. H. Kim. 2003. Transepithelial migration of neutrophils in response to leukotriene B4 is mediated by a reactive oxygen species-extracellular signal-regulated kinase-linked cascade. J. Immunol. 170: 6273-6280. [Abstract/Free Full Text]
  49. Tanimura, S., K. Asato, S. H. Fujishiro, M. Kohno. 2003. Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: down-regulation of matrix metalloproteinase-3/-9/-14 and CD44. Biochem. Biophys. Res. Commun. 304: 801-806. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Sharabi and E. Mozes
The Suppression of Murine Lupus by a Tolerogenic Peptide Involves Foxp3-Expressing CD8 Cells That Are Required for the Optimal Induction and Function of Foxp3-Expressing CD4 Cells
J. Immunol., September 1, 2008; 181(5): 3243 - 3251.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. P. Singh, A. La Cava, and B. H. Hahn
pConsensus Peptide Induces Tolerogenic CD8+ T Cells in Lupus-Prone (NZB x NZW)F1 Mice by Differentially Regulating Foxp3 and PD1 Molecules
J. Immunol., February 15, 2008; 180(4): 2069 - 2080.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
U. Sela, M. Dayan, R. Hershkoviz, O. Lider, and E. Mozes
A Peptide That Ameliorates Lupus Up-Regulates the Diminished Expression of Early Growth Response Factors 2 and 3
J. Immunol., February 1, 2008; 180(3): 1584 - 1591.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sela, U.